Hemophilia A is a genetic disease where disorder or deficiency of FVIII leads to reduction of the blood coagulation activity, resulting in bleeding in the bowels or at the joint, etc. For treatment of patients suffering from hemophilia A, supplemental treatment of FVIII has been carried out. In about 10% (8 to 15%) out of the patients who received supplemental treatment, however, induction of an antibody to FVIII has been observed. This antibody, called “anti-FVIII inhibitor antibody”, makes supplemental treatment with the blood coagulation factors ineffective and hemostatic management of the hemophilia patients extremely difficult.
Elucidation of nature of the anti-FVIII inhibitor antibody and its production mechanism would enable inhibiting or regulating production of the inhibitor antibody. Up till the present, there has been research using an anti-FVIII inhibitor antibody (polyclonal antibody) purified from the sera of hemophilia patients having anti-FVIII inhibitor antibodies or a mouse anti-FVIII inhibitor antibody (mouse monoclonal antibody) to thereby provide much information. As to cloning of anti-FVIII inhibitor antibody from patients, however, there has almost been no report.
On the other hand, epidemiological study has shown that hemophilia patients exhibited low occurrence of arteriosclerosis such as myocardial infarction, in particular, significantly low occurrence of lethal myocardial infarction (Br. J. Haemotol. 1990, 75, 525–530; Lancet 1995, 345(8943) 152–155); and that an increased FVIII level in plasma may possibly be a risk factor for arterial infarction or arteriosclerosis (Semin. Hematol. 1997, 34, 171–187; Thromb. Res. 1997, 84, 359–362).
Thus, it is expected that, if lowered blood coagulation could be maintained by using an anti-FVIII inhibitor antibody, this would contribute to prevention and treatment of arterial infarction and arteriosclerosis. In particular, an anti-FVIII antibody that specifically recognizes activated FVIII free from von Willebrand factor (hereinafter also referred to as “vWF”) exhibits much long clearance in blood as failing to complex with FVIII usually bound with vWF in blood, and hence can be an excellent anti-thrombotic drug in which lowered blood coagulation may be maintained for a long period of time with a single administration and bleeding or other adverse side effects can be much reduced.
There have already been numerous attempts to prepare mouse monoclonal antibodies to FVIII in many laboratories including those prepared by the present inventors and so a number of monoclonal antibodies to FVIII have been produced. Some of them have been proved to inhibit the FVIII activity and to behave as an anti-FVIII inhibitor antibody. With these mouse monoclonal antibodies, numerous results have been obtained including analysis of regions that inhibit the FVIII activity, etc.
A completely human anti-FVIII inhibitor antibody, however, has scarcely been prepared with success although it has been attempted over more than ten years in many laboratories with the hybridoma technique (human/mouse or human/human hybridoma technique) or the EB transformation technique, using lymphocytes from patients having anti-FVIII inhibitor antibodies.
In 1998, Marc G. Jacquemin et al. reported first establishment of the method for preparing a completely human anti-Factor VIII inhibitor antibody using the EB transformation technique (Blood, 92(2), 1998, 496–506). They established BO2C11 antibody which was thought to inhibit binding of FVIII with vWF or with phospholipids and to recognize the C2 domain of FVIII. VH and VL genes of this antibody were derived from DP5 fragment and VK3, respectively. They also succeeded in establishment of clones producing anti-Factor VIII inhibitor antibodies to the C1 domain with the similar method and reported that the antibodies are derived from DP64 and VK3 (Blood, 95, 2000, 156–163). Up till the present, it is only Marc G. Jacquemin et al. who has ever established a clone producing such a human-derived anti-FVIII inhibitor antibody with the hybridoma technique (human/mouse or human/human hybridoma technique) or the EB transformation technique.
On the other hand, the antibody display phage technology reported by J. D. Marks et al. (J. Mol. Biol, 222, 581–597, 1991) is advantageous in that an antibody repertoire from lymphocytes from patients can be screened in a shorter period of time and in more clones than the conventional hybridoma technique or the EB transformation technique. In recent years, there has been an attempt to use this antibody display phage technology for isolating the inhibitor antibody.
W. H. Ouehand et al. reported in the International Congress on Thrombosis and Hemostasis, 1995, June (Israel) (1995, ISTH) that they cloned a human anti-FVIII single-chain antibody (scFv) from human antibody display phage libraries from healthy donors and a library of synthetic human phage antibodies. J. Davis et al. also reported in the International Congress on Thrombosis and Hemostasis, 1997, June that they cloned a human anti-FVIII single-chain antibody (scfv) from human antibody display phage libraries wherein VH genes were derived from hemophilia patients and VL genes were from healthy donors. In these reports, however, the human antibody display phage libraries were constructed in a synthetic or semi-synthetic manner in which not both of VH and VL was derived from hemophilia patients. Moreover, although the clones obtained in these reports bound to FVIII as shown by ELISA, it was not proved whether the clones inhibited the coagulation activity of FVIII.
In 2000, E. N. van den Brink et al. reported that they constructed an antibody display phage library in which VL chain genes were derived from healthy donors and VH chain genes were from patients having the inhibitor antibodies and isolated four clones reactive with FVIII, of which two clones were reactive with the C2 domain of FVIII whereas the other two were reactive with the A2 domain of FVIII [van Den Brink, Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes (Blood, 2000; 95: 558–563); Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain (Blood, 2000; 96: 540–545)].
It has been reported that the C2 domain of FVIII was involved in binding with vWF or with phospholipids and was a principal recognition region of many anti-FVIII inhibitor antibodies present in blood from patients (Healey, J. F. et al., Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII, Blood, 1998, 92, 3701–3709).
According to the study by van den Brink et al., the antibody fragments to the C2 domain obtained thereby did not have an inhibitor activity to FVIII. Accordingly, a human anti-FVIII antibody with the inhibitor activity has not yet successfully been isolated, hence failing to provide sufficient information.
The reason why a human anti-FVIII antibody with the inhibitor activity could not successfully be isolated with the antibody display phage technology was presumed to be that genes of VL chain of antibody fragments were not derived from patients and that there might be some problems in the screening procedure.